Without limiting the scope of the invention, its background is described in connection with aptamers generally and traditional aptamer generation methodologies.
Aptamers are structurally distinct RNA and DNA oligonucleotides (ODNs) that can mimic protein-binding molecules and exhibit high (nM) binding affinity based on their unique secondary three-dimensional structure conformations and not by pair-wise nucleic acid binding. Aptamers can be selected via high-throughput in vitro methods to bind target molecules. Aptamers are thus emerging as viable alternatives to small molecules and antibody-based therapies in the field of drug development.
Aptamers are typically approximately 1/10th the molecular weight of antibodies and yet provide complex tertiary, folded structures with sufficient recognition surface areas to rival antibodies. However, aptamers achieve their selectivity through a very limited repertoire of functional groups—the sugar phosphate backbone and 4 bases. In contrast antibodies use all 20 amino acids with a full range of chemical substituents including positively-charged, sulfhydryl, hydrophobic sidechains, etc. Aptamers are polyanions, potentially limiting their affinity towards the full diversity of proteins. It is often difficult to select an aptamer targeted to very acidic proteins because there are no cationic groups to neutralize anionic surfaces on the protein. While oligonucleotide agents show therapeutic promise, various pharmacological problems must be overcome. High sensitivity to nuclease digestion makes oligonucleotide agents unstable and thus impracticable for in vivo administration by either intravenous or oral routes.
In fact, a diverse range of modifications at all possible modification sites of an oligonucleotide have been reported for enhancing oligonucleotide drug properties including in vivo stability. These include alterations of linkages (backbones), heterocycles, carbohydrates, and connection and conjugation sites, as well as the complete removal of the sugar-phosphate backbone. See, e.g. Yang X; Li N; Gorenstein D G Expert Opin Drug Discov 6 (2011) 75-87; Brody E N, et al. Expert Rev Mol Diagn 10 (2010) 1013-22; Keefe A D, Cload S T Current Opinion in Chemical Biology 12 (2008) 448-456.
Certain of the present inventors have developed thiophosphate-backbone modified aptamers (“thio” aptamers) as specific protein-binding reagents that are endowed with nuclease resistance. See, e.g. Yang, X. et al. Nucleic Acids Research 30 (2002) e132; Yang, X. et al. Nucleic Acids Research 31 (2003) e54. Oligonucleotides with high monothio- or dithiophosphate backbone substitutions enhance the specificity and affinity of these agents for the desired protein target and also enhance the nuclease stability.
However effort to combine the best attributes of antibodies, small molecules and aptamers has remained elusive. Selection of aptamers by the classical iterative selection-amplification method followed by post-selection modification has been disappointing because the modifications affect the three dimensional structure of the aptamer, which is the basis of its ability to bind to the target by which it was selected. It has been shown that certain substituents can be introduced into the bases of the oligonucleotides to provide additional functionalities. For instance, the 5-position of dU can be replaced with a range of substituents (X) and still allow Taq and other polymerases to amplify the selected sequences. Thus, with the appropriate 5-X-dUTP, it is possible to amplify a selected sequence during the in vitro iterative SELEX scheme and create a large initial random library (1014 different sequences), then select a subset that binds to the target protein, amplify and repeat this cycle—often 10-15 cycles are required. The problem is that every 5-X-dU position will have the same modified X-substituent.
From the foregoing it is apparent there is a need in the art for robust methods that allow the selection of modified aptamers having desired chemical substituents. The invention described herein provides novel methods for achieving this end.